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Phyllanthus muellerianus (Kuntze) Excell (family Euphorbiaceae) stem bark methanol extract inhibited the growth of Clostridium sporogenes and Streptococcus pyogenes, responsible for gas gangrene and suppurative and non suppurative diseases, respectively. After the HPLC fingerprint acquisition a bioguided fractionation of the defatted methanol extract allowed the isolation of six fractions whose activity was evaluated against the two pathogen bacteria. A further purification of the most active fraction afforded a pure compound responsible for the very interesting inhibitory activity against C. sporogenes and S. pyogenes (MIC 0.91 μM, MIC 3.64 μM). (1)H NMR and MS analytical techniques allowed the identification of the bioactive as Nitidine; this quaternary ammonium alkaloid was observed in the genus Phyllanthus for the first time. A study on Nitidine counter ion, performed using energy dispersive spectroscopy (EDS) coupled with scanning electron microscopy (SEM) was also carried out.

Aqueous, methanolic and ethyl acetate extracts of Piptadeniastrum africanum (Hook f.) Brenan stem bark were tested against fungi and representatives of Gram-positive and Gram-negative bacteria. Aqueous and methanolic extracts showed a maximum activity against the fungus Pyricularia grisea, a weak activity against the bacteria Staphylococcus aureus and Streptococcus mutans, while no activity was observed against Gram-negative bacteria. Ethyl acetate extract resulted inactive against all the tested microorganisms. Phytochemical investigations of the most active methanolic extract highlighted the presence of tannins and saponins. After purification by means of Sephadex LH-20 and RP-18 the tannin fraction resulted more active against bacteria, while the saponin fraction showed a pronounced activity against Pyricularia grisea. Five different strains of Pyricularia grisea were used and results show that the saponin fraction is promisingly active on the whole population (MIC and MFC: 0.03–0.06 mg mL−1). The saponin fraction fungicidal activity was similar to that of Flutriafol (0.01–0.04 mg mL−1), a commercially available fungicide, indicating that P. africanum stem bark extract can be a good candidate for the control of Pyricularia grisea, the most diffused rice blast disease fungus of rice.

The plant kingdom is an endless source of chemically diverse bioactive compounds, which are used in traditional folk medicine for the treatment of a wide array of diseases. A previous investigation on the bioactive phytocomponents present in the methanol extract of Phyllanthus muellerianus (PMME) demonstrated an interesting activity against C. sporogenes (MIC= 100 µg/ml) and S. pyogenes (MIC= 300 µg/ml) [1], which supported the traditional use of the extract by local populations in Cameroon. Looking for activity beyond the claimed traditional use [2], PMME was evaluated on human cholinesterase enzymes, namely acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) as selected targets for Alzheimer’s disease (AD) treatment. Indeed AChE inhibitors derived from natural products (galanthamine and rivastigmine) are currently licensed to alleviate cognitive symptoms in dementia. Due to the limited available pharmacological treatments for AD, extensive research has been directed towards the identification of other AChE inhibitors, arising from the plant kingdom. A rational basis for our investigation on the bark extracts of Phyllanthus muellerianus is related to a study carried out in 2007 by Joshi e Parle, in which the antiamnesic potential for Phyllanthus amarus in mice was demonstrated [3]. Anticholinesterase activity was in vitro evaluated by Ellman’s assay [4] using recombinant human AChE and BuChE from human serum. Due to the interesting activity found for the de-fatted PMME (% inhibition at 100 g mL-1 on hAChE = 52.6 ± 0.6, on hBuChE = 70.1 ± 3.1%, n=4), PMME was fractionated by flash chromatography affording six fractions (PMF1-6); PMF1, PMF2 and PMF4 significantly inhibited human cholinesterases, thus they were further purified by a RP-18 solid phase extraction. The bio-guided fractionation allowed the identification of three active phytocomponents, with different selectivities against the two ChEs. Two out of the three bioactives have been isolated so far: compound A from PMF1 and compound B from PMF4, both alkaloids. Compound A showed to be a hBuChE selective inhibitor (IC50 = 44.8 ± 3.0 g mL-1 and IC50 = 382 ± 15 g mL-1, for hBuChE and hAChE, respectively) while compound B showed comparable inhibitory potencies against both enzymes (IC50 = 5.45 ± 0.20 g mL-1 and IC50 = 12.6 ± 0.4 g mL-1, for hAChE and BuChE, respectively). Compared to the commercially available AD drug galanthamine, compound B is one order of magnitude less active on hAChE (galanthamine IC50 = 0.6 g mL-1), representing, however, a potential new natural scaffold to undergo towards optimization. The chemical structure of the two isolated alkaloids is under investigation by 13C and 1H-NMR. Moreover, the isolation of compound C in suitable amount for the evaluation of the biological profile and its structural elucidation has being carried out. References [1] G. Brusotti, I. Cesari, G. Frassa, P. Grisoli, C. Dacarro, G. Caccialanza, J. Ethnopharmacol., 2011, 35, 797–800. [2] R. Brisson, Etudes pygmées, SELAF n 376, Ed Peeters, 1999, Paris. [3] H. Joshi and M. Parle, African Journal of Biomedical. Research., 2007, 10, 165–173. [4] G.L. Ellman, K.D. Courtney, V. Jr. Andres, R.M. Feather-Stone, Biochem. Pharmacol., 1961, 7, 88-95

The phytochemical research based on ethnopharmacology is considered an effective approach in the discovery of novel chemicals entities with potential as drug leads. Plants/plant extracts/decoctions, used by folklore traditions for treating several diseases, represent a source of chemical entities but no information are available on their nature. Starting from this viewpoint, the aim of this review is to address natural-products chemists to the choice of the best methodologies, which include the combination of extraction/sample preparation tools and analytical techniques, for isolating and characterizing bioactive secondary metabolites from plants, as potential lead compounds in the drug discovery process. The work is distributed according to the different steps involved in the ethnopharmacological approach (extraction, sample preparation, biological screening, etc.), discussing the analytical techniques employed for the isolation and identification of compound/s responsible for the biological activity claimed in the traditional use (separation, spectroscopic, hyphenated techniques, etc.). Particular emphasis will be on herbal medicines applications and developments achieved from 2010 up to date.

Ethnopharmacological relevance Diospyros bipindensis (Gurke) stem bark is used in Cameroon by Baka Pygmies for the treatment of respiratory disorders. Aim of the study To assess the anti-inflammatory, antibacterial and antioxidant properties of constituents from the bark extracts through bioassay-guided fractionation. Materials and methods The anti-inflammatory activity of extracts, fractions and pure compounds was assessed through the inhibition of the pro-inflammatory mediator nuclear factor-kappa B (NF-κB) transcriptional activity and nitric oxide (NO) production. DPPH, ABTS and ORAC assays were used for determining the antioxidant properties. The activity against Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli and Klebsiella pneumoniae , was evaluated on the basis of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) by the macrodilution method. Results The water extract showed antimicrobial activity against S. pneumoniae (MIC: 300 μg/ml) and S. pyogenes (MIC: 300 μg/ml). The dichloromethane extract efficiently inhibited NF-κB transcriptional activity and NO production and exhibited significant antioxidant activity in the ORAC assay. An interesting activity was also found against S. pneumoniae (MIC: 200 μg/ml), S. aureus (MIC: 400 μg/ml) and S. pyogenes (MIC: 200 μg/ml). The phytochemical investigation of the dichloromethane extract afforded plumbagin, canaliculatin, ismailin, betulinic acid and 4-hydroxy-5-methyl-coumarin as the main constituents. Plumbagin and ismailin were found to be responsible for the main biological activities observed. Conclusions These results may provide a rational support for the traditional use of Diospyros bipindensis stem bark in the treatment of respiratory disorders, since the anti-inflammatory, antimicrobial and antioxidant compounds isolated from the dichloromethane extract were also present in the traditional water extract.

Ethnopharmacological relevance Phyllanthus muellerianus (Kuntze) Excel (family Euphorbiaceae) stem bark is used in Cameroon by Baka pygmies as a remedy for wound healing and tetanus. Aim of the study To characterize the chemical composition and to evaluate the antimicrobial properties of the essential oil of the plant. Materials and methods The essential oil was extracted from the stem bark by dynamic head space and by hydrodistillation and characterized by GC and GC-MS analyses. The antimicrobial activity was evaluated on the basis of the minimum inhibitory concentration (MIC) and the minimum bactericidal–fungicidal concentration (MBC–MFC) by the micro and macrodilution methods. The following bacteria and fungi were used: Clostridium sporogenes ATCC 3584, Staphylococcus aureus ATCC 6538, Streptococcus mutans ATCC 25175, Streptococcus pyogenes ATCC 19615, Escherichia coli ATCC 10536, Candida albicans ATCC 10231, Candida albicans LM 450, Trichophyton mentagrophytes LM 230, Trichophyton rubrum LM 237, Microsporum canis LM 324. Results The hydrodistillation afforded 0.06% (dry weight basis) of pale yellow oil. Thirty-eight compounds representing 90.69% were identified. The major component (36.40%) was found to be (E)-isoelemicin, identified by comparison of its 1H-NMR experimental data, with literature data. The oil showed good antibacterial activity against Clostridium Sporogenes, Streptococcus mutans and Streptococcus pyogenes with MIC ranging from 13.5 to 126 μg/ml. A weak antifungal activity (MIC 250 μg/ml) was found against Trichophyton rubrum, only. Conclusions The antimicrobial activity and the chemical composition of Phyllanthus muellerianus stem bark essential oil are reported for the first time.

Ethnopharmacological relevance The plants of the genus Phyllanthus (Euphorbiaceae) are widely distributed in most tropical and subtropical countries, and have long been used in folk medicine to treat several diseases. Particularly, Phyllanthus muellerianus (Kuntze) Excell, commonly called “mbolongo” in Cameroon, is used by pygmies baka as a remedy for tetanus and wound infections. Aim of the study To investigate the antimicrobial properties of Phyllanthus muellerianus (Kuntze) Excell (family Euphorbiaceae) stem bark used in Cameroon by baka pygmies as a remedy for wound healing and tetanus. Materials and methods Aqueous and methanol extracts with and without defatting treatment, were prepared and their activity against Clostridium sporogenes ATCC 3584, Staphylococcus aureus ATCC 6538, Streptococcus mutans ATCC 25175, Streptococcus pyogenes ATCC 19615, Escherichia coli ATCC 10536, Candida albicans ATCC 10231, was evaluated on the basis of the minimum inhibitory concentration (MIC) and the minimum bactericidal-fungicidal concentration (MBC-MFC) by the macrodilution method. Results Water extract showed a weak activity against Clostridium sporogenes (MIC 900 μg/mL) and resulted inactive at the tested concentrations against all the other microorganisms. The defatted methanol extract, inactive against Staphylococcus aureus , Escherichia coli , Candida albicans , exhibited a very interesting activity against Clostridium sporogenes and Streptococcus pyogenes (MIC 100 μg/mL and 300 μg/mL, respectively), which seems to validate the use of this plant in pygmies traditional medicine for the treatment of tetanus and wound infections. The activity found against Streptococcus mutans (300 μg/mL), aetiological agent of caries, may suggest a possible use of this plant as natural remedy to prevent dental diseases. Conclusions The activity against streptococci and Clostridium sporogenes ATCC 3584, showed by stem bark extracts of Phyllanthus muellerianus , traditionally used by baka pygmies to treat wound infections and tetanus, is reported for the first time.

A comparative study of some physico-chemical properties of alkaline phosphatase of a human cell line, the EUE, with high level of enzyme and one of its clonal derivatives the E6D, with low activity, has been carried out. Electrophoretic analysis reveals a multiple banding pattern within each line and qualitative differences between the two lines. The alkaline phosphatase activity of the E6D cell extracts is almost completely inhibited by 5 × 10−2 M inorganic phosphate while in the EUE the enzymic activity is reduced to one third under these conditions. The enzymes of the two lines show also a different thermostability which is not referable to extrinsic factors, as demonstrated by mixing experiments. The time course of heat inactivation at 70°C suggests molecular heterogeneity in each line, and a prevalence of a thermostable fraction in the cells with low activity and a thermolabile one in those with high enzymic levels. A rough estimate of inactivation constants does not rule out the possibility that the molecular species in the two lines are the same but in different proportions. The cytological analysis confirms the relationship between the number of small acrocentric chromosomes and alkaline phosphatase levels. The significance of the biochemical data in relation to the proposed model of a gene dosage effect is discussed.